Casein gene expression in murine mammary gland explants has an absolute requirement for insulin, glucocorticoid and prolactin. The requirement for insulin is independent of the hormone's cell-maintenance properties. Of all the factors tested only anti-insulin receptor antibodies can effectively substitute for insulin. In the presence of cell-maintenance agents such as MSA or FCS, casein gene transcripts are expressed equally well regardless of whether insulin or prolactin is added last to the culture.